Technical Abstract:
Background: Escherichia coli biofilm formation is dependent on curli fimbriae and cellulose, and the expression of both varies among Shiga toxin-producing E. coli (STEC). Curli and cellulose expression are often identified by their affinity for Congo red dye (CR) but media composition and incubation temperature can affect dye affinity, imposing limitations on this method. We compared CR binding by various STEC on 2 different growth media at 3 incubation temperatures. We also used curli and cellulose mutant strains, and cellulose staining to differentiate cellulose and curli contributions to CR binding by different strains and serotypes.
Methods: Isolates of serotype O157:H7, STEC serogroups O26, O45, O103, O111, O113, O121, and O145, and isogenic mutant strains of selected serotypes were spotted on Congo red indicator (CRI) agar and T-medium agar and incubated at 25ºC, 30ºC, and 37ºC for 48 h. Mutations were constructed in the curli (csgBA or csgDEFG) and cellulose (bcsABZC) operons using RedET recombination. Calcofluor was added to T-medium agar to detect cellulose.
Results: Most STEC strains show higher curli expression and CR affinity on T-medium agar compared to CRI. However, there were distinct differences between O157:H7 and most non-O157 STEC in temperature control of CR affinity. Cellulose contributed little to CR affinity in serotype O157:H7 but interacted with curli in the non-O157 STEC producing variable effects on CR affinity. Medium-dependent cellulose expression was shown in the tested O45 strains.
Conclusions: Media composition and temperature requirements for maximum CR affinity differ among STEC serotypes and should be considered when screening for curli and cellulose.